Does fetal hemoglobin inhibit the malarial parasite <i>Plasmodium falciparum</i>?
Authors: Natasha M. Archer, Geoffrey Pasvol, Iain Wilson, Manoj T. Duraisingh
Journal: American Journal of Hematology (2022)

The human malaria parasite Plasmodium falciparum interacts with numerous polymorphic molecules in its host red blood cell (RBC), which presents difficulties in examining the effect of single RBC determinants on parasite infection. A good illustration is the story of fetal hemoglobin (HbF). First explored by two of the authors nearly five decades ago, the question of whether HbF inhibits invasion and/or growth continues to arise due to acquisition of epidemiologic data suggesting an interaction between HbF and sickle hemoglobin (HbS) that may influence malarial infection.1, 2 Here, we detail the studies investigating HbF's effect on malarial infection, highlighting the complexities associated with interpretation and providing suggestions of how to investigate further polymorphisms in parasitized RBCs from natural populations. In foundational studies using experimental culture in vitro of infected mixed umbilical cord and HbAA cells, Pasvol et al. demonstrated that P. falciparum has a predilection for younger RBCs.3 This was done by analyzing blood smears from patients and cultures treated with a modification of the acid-elution-differential staining method of Kleihauer et al., a procedure that distinguishes parasitized HbF cells (F-cells) from normal (HbAA) cells since HbF is resistant to elution at pH 3.5.4 Thus, cells with differing hemoglobins could be tested under identical conditions rather than in parallel cultures. A higher percentage of F-cells, the younger and more metabolically active cells in the cord and HbAA blood mixture, were invaded by parasites. In addition, younger stages of parasites were seen in F-cells in the single cord blood sample used. Compared to the complete stalling of growth in sickle cell trait RBCs,5, 6 the growth delay was slight but significant. Lastly, Pasvol et al.3 confirmed that P. falciparum preferentially invades younger RBCs and not F-cells in infant blood samples. The younger cells in infant blood, that is, those that are HbA dominant, were more often invaded in up to two cell cycles. While these studies clearly demonstrated an effect of age and not HbF on parasite invasion, a slight but measurable effect of HbF on growth, was noted. A subsequent study using mixtures of HbF and HbA dominant RBCs again confirmed that invasion was not influenced by HbF but by age of the RBCs.7 In addition to the cord-adult blood mix and blood of infants aged 3–6 months used in the above experiments, invasion into blood of heterozygotes of African Hereditary Persistence of Fetal Hemoglobin (HPFH) and a mix of blood of homozygotes for British HPFH and adult HbAA blood were studied. Once again, the younger cells (cord and HbAA RBCs) in the first two samples, respectively, were preferentially invaded. In the two HPFH samples, in which all RBCs were of the same age, there was no preference for either HbF or HbA dominant RBCs. Pasvol, Weatherall and Wilson also published another set of studies highlighting the preferential invasion of reticulocytes by P. falciparum.8 In these experiments, centrifugation was followed by isolation of RBCs from the top, middle and bottom of Wintrobe tubes. RBCs were then stained for reticulocytes with brilliant cresyl blue and counterstained for parasites with Giemsa's stain. Compared to normocytes, a higher parasitemia was found in reticulocytes and the top fraction that contains them. Wilson et al. also showed that the size of parasites in RBCs from a single culture of cord and adult blood were smaller in the F-cells.9 These studies again showed that HbF had no influence on invasion but suggested an influence on growth. In these early studies, the focus was primarily the preferential parasitic invasion of younger cells with brief comments regarding delayed parasite development in F-cells. As a result, other studies10, 11 were conducted particularly focused on a mechanism for the HbF induced development delay. Friedman et al. could only show inhibition of parasite growth in HbF dominant RBCs (unspecified RBC type) in vitro in glutathione reduced media (medium 199), the same medium used in the Pasvol and Wilson studies. In RPMI (Roswell Park Memorial Institute) media, normal growth was observed. This suggested a mechanism of oxidant stress.10 Later, Shear et al., demonstrated a protective effect of HbF in vivo in a transgenic rodent malaria model with humanized alpha and gamma globin chains.11 When infected with P. yoelii (17XL), transgenic mice had a lower peak parasitemia, cleared their infection by day 24, and survived; control mice died between days 11 and 13. In transgenic mice with a P. chabaudi adami infection, the parasite parasitemia rose quickly but was also rapidly cleared. The infection was not reversed by splenectomy. In biochemical studies, Shear et al. also showed that human HbF was digested half as well as human HbA by a recombinant parasite hemoglobinase (Plasmepsin II) and proposed stability of the HbF tetramer as a potential mechanism for protection by HbF. The differential growth in the two media found by Friedman and the lack of specific details regarding the RBCs of the transgenic mice and their relevance to polymorphic human populations, as well as the purely biochemical data collected in Shear et al., still left questions regarding HbF's detrimental influence on growth. Therefore in 2011, Amaratunga et al. re-examined this finding and showed no difference in development in cultures of six cord blood samples and one HPFH compared to HbA dominant blood using five different parasite lines (7G8, FVO, GB4, MC/R+, TM284). Flow cytometry was employed to assess development, measuring DNA replication in growth, compared to the previous work that relied on subjective morphological assessment. Amaratunga and colleagues however did not assess for age of RBCs, nor did they follow cultures beyond 40 h (a noted limitation due to known RBC aging observed in in vitro culture) so a growth delay from the trophozoite to the schizont stage was still possible. Amaratunga et al. also demonstrated a reduction in PfEMP-1 surface expression, rosetting, and adherence to microvascular endothelial cells and monocytes by parasitized cord and HPFH RBCs compared to HbA dominant RBCs. They proposed a new protective mechanism of HbF, namely reduced cytoadherence.12 Considering the finding of no difference in parasite growth in cord and HbA dominant RBCs by Amaratunga et al., Sauerzopf et al. investigated the potential role of maternal immunoglobulins in limiting growth.13 They too found no difference in parasite growth in cord and HbA dominant RBCs using the HRP2-assay. However, when non-malaria-exposed donor plasma was used compared to maternal or cord blood plasma (presumably exposed); growth in the plasma from malaria exposed individuals was reduced. Cyrklaff et al. also found no effect on parasite multiplication rate and hemozoin levels up to 12 cycles but demonstrated impaired actin remodeling in HbF RBCs14 by comparing cryo-tomographic images of FCR3CSA in adult, cord, sickle cell trait, and hemoglobin C trait erythrocytes. The shorter actin filaments in non-HbAA RBCs suggested a dysfunctional cytoskeleton. In addition, Maurer's clefts and knobs were malformed and the amount of VAR2CSA presented on the red cell surface was reduced. These later studies all pointed toward reduced cytoadherence and not growth inhibition as a mechanism of HbF protection. To elucidate the potential effect of HbF in patients with sickle cell disease treated with hydroxyurea, Archer et al. also compared overall parasite growth and proliferation in umbilical cord blood and heterozygote African HPFH versus adult blood (HbAA only) using cell sorting and the cultured parasite line Pf3D7 IG06. No significant difference in parasite intraerythrocytic development and parasite multiplication rate (PMR) was noted in HbF and HbA dominant RBCs,15 in contrast with the significant growth defect seen in sickle cell trait RBCs in low oxygen.6 In reviewing these various data, the authors, recommend the following when considering future studies of HbF's effect on malaria invasion, development, and proliferation: (1) RBC age is likely a confounder given the numerous changes experienced by the RBC during erythropoiesis. To avoid age differences in RBC, HPFH in the absence of other hemoglobinopathies or isogenic RBC lines should be used; (2) While cultured parasites are easier to obtain and manipulate, where possible, primary field isolates should be used to confirm findings; (3) Given the polygenicity of HbF with other genes,16, 17 it is important that large sample sizes be used to establish small size effects. When the number of RBC blood samples is finite, single cell isolation of HbF positive and negative cells as done by Pasvol et al. via acid-elution and more recently by flow cytometry should be employed. In summary, the effect of HbF on parasite growth is at best relatively small and may only be significant in the setting of a high oxidant stress environment. However, in the context of polygenic effects of protection against lethal malarial infection, HbF might play a role for better or for worse. Future studies should consider these possibilities. NMA is supported by research funding from National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) 1K08DK123386-01A1.MTD is supported by funding from National Institute of Immunology and Infectious Diseases (NIAID) R01AI40751 and U19AI089688. The authors declare that they have no conflict of interest. Data sharing is not applicable to this article as no new data were created or analyzed in this study. Data sharing is not applicable to this article as no new data were created or analyzed in this study.